Natural Product Research
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3.1. Plant material
The fruit of T. terrestris L. was from the Ji-Lin province of China and identified
by a company in Nanjing. The voucher of this plant material is on file in our laboratory.
3.2. Extraction and isolation
The dried fruits of T. terrestris (1.8 kg) were thrice extracted with 70% EtOH. The
solutions were combined and evaporated to dryness to give a crude extract of 450.6 g. The
extract was then subjected to D101 resin column chromatography eluted gradiently with
H2O/EtOH solutions to give H2O fraction (244.7 g), 20% EtOH fraction (56.2 g), 70%
EtOH fraction (66.7 g) and 90% EtOH fraction (83.4 g), respectively. The 20% EtOH
fraction (56.2 g) was then subjected to silica gel column chromatography (Wako gel C-300,
Wako pure Chemical Industry Ltd., 700 g, 12 ꢃ 17 cm, eluted with CHCl3 : MeOH : H2O
(8 : 2 : 0.2; 7 : 3 : 0.5)) to give nine portions. The fractions were combined on the basis of
TLC with Ehrlich’s spray reagent. The fourth portion was then separated by silica gel
column chromatography and eluted with CHCl3 : MeOH 100 : 0–0 : 100) into fractions 1–7,
and fraction 4 was further purified by HPLC (Senshu pak PEGASIL ODS Al,
10 ꢃ 250 mm, MeOH : H2O (70 : 30), flow rate 4 mL minꢁ1; UV detector at 254 nm) to
give five furostanol saponins: compound 1 (504 mg), compound 2 (10 mg), compound 3
(15.4 mg), compound 4 (15.1 mg) and compound 5 (20.3 mg).
3.3. Structure and identification
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Compound 1: White power, [m.p.] 215–217ꢂC, ½ꢁꢀD –38.2ꢂ(H2O, c 0.68), [HR-TOF-MS]
1335.6185 [M þ Na þ H]þ. ESI-MS: 1311.4 [M ꢁ H]ꢁ, 1335.3 [M þ Na]þ. For 1H and 13C-
NMR (pyridine-d5) data, see Tables 1 and 3, respectively.
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Compound 2: Colourless crystal, [m.p.] 220–222ꢂC, ½ꢁꢀD –25.2ꢂ (H2O, c 0.56), [HR-TOF-
MS] 1381.6250 [M þ Na]þ, ESI-MS: 1357.6 [MꢁH]ꢁ, 1381.6 [M þ Na]þ. For 1H and 13C-
NMR (pyridine-d5) data, see Tables 1 and 3, respectively.
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Compound 3: White power, [m.p.] 214–216ꢂC, ½ꢁꢀD ꢁ29.6ꢂ (H2O, c 0.28), [HR-TOF-MS]
1
1367.6095 [M þ Na]þ, ESI-MS: 1343.6 [M ꢁ H]ꢁ, 1367.5 [M þ Na]þ. For H and 13C-
NMR (pyridine-d5) data, see Tables 1 and 3, respectively.
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Compound 4: White power, [m.p.] 230–232ꢂC, ½ꢁꢀD –39.3ꢂ (H2O, c 0.50), ESI-MS: 1329.6
[M ꢁ H]ꢁ, 1353.6 [M þ Na]þ. For 1H and 13C-NMR (pyridine-d5) data, see Tables 1 and 3,
respectively.
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Compound 5: Colourless crystal, [m.p.] 225ꢁ227ꢂC, ½ꢁꢀD –32.5ꢂ (H2O, c 0.48), ESI-
MS: 1343.8 [MꢁH]ꢁ, 1367.7 [M þ Na]+. For H and 13C-NMR (pyridine-d5) data, see
1
Tables 1 and 3, respectively.
3.4. Acid hydrolysis and GC-MS
Each glycoside (5 mg) was heated in a sealed tube with 5mL of aqueous 2 M HCl at 100ꢂC
for 2 h. The aglycone was extracted with dichloromethane, and the aqueous residue was
evaporated under reduced pressure. Then, 1 mL pyridine and 2 mg NH2OH ꢄ HCl were
added to the dry residue, and the mixtures were heated at 100ꢂC for 1 h. After the reaction